Project 2: Sodroski ? Impact of Env conformational regulation on HIV-1 sensitivity to small-molecule entry inhibitors SUMMARY The human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer binds to host receptors, CD4 and CCR5/CXCR4, and mediates the entry of the virus into the target cell. As a result of receptor binding, the Env trimer undergoes large-scale conformational changes from the unliganded state (State 1) to more ?open? conformations (States 2 and 3). Small-molecule inhibitors of HIV-1 entry bind the Env trimer and either block conformational changes or prematurely trigger non-productive conformational changes. Thus, small- molecule entry inhibitors serve as useful probes of Env trimer conformation. In this project, we will utilize crosslinking and mass spectrometry to evaluate the conformation of HIV-1 Env in the absence and presence of small molecules. We will evaluate the impact of natural HIV-1 variation on virus sensitivity to CD4-mimetic compounds. We will investigate the Env binding site and mechanism of action of another small-molecule HIV-1 entry inhibitor, MF275. MF275 inhibits infection of some HIV-1 strains, but enhances the infection of CD4-negative target cells by other HIV-1 strains. The CD4-mimetic compounds and MF275 bind to distinct sites on the HIV-1 Env trimer, and prematurely activate Env by driving it from State 1 into States 2 and 3. In addition to these more focused aims, we will evaluate the Env conformational effects and antiviral potency, breadth and specificity of novel HIV-1 entry inhibitors developed in Projects 3 and 4 of this Program. An understanding of the impact of HIV-1 Env conformational variation on the antiviral activity of small-molecule inhibitors will assist efforts to improve their efficacy.